Changes in the membrane potential of cells are at the basis of many fundamental physiological processes, including heartbeat, brain function, visual and olfactory transduction, and muscle contraction. Cells are often using the electrical signaling to communicate with each other and to change their behavior according to external clues.
The plasma membrane ensures its structural integrity of a cell, and physically separates intracellular compartments from extracellular surroundings. An electric field (E-field) gradient is present across the cell membrane due to a) substantial differences in ion compositions between intracellular and extracellular compartments, and b) insulating physical properties of phospholipid bilayers comprising the cell membrane. At rest, the inside of cells is more negatively charged than the outside, resulting in the resting membrane potential in the range from −10 to −80 mV in different types of cells. Changes in the membrane potential can be triggered by electrical and/or chemical signals. Specifically, these signals lead to opening of voltage-gated or ligand-gated ion channels, resulting in changes in membrane permeability, redistribution of ions on intracellular and extracellular sides of the plasma membrane, and, finally, changes in the membrane potential.
To control the membrane potential is to control the activation state of a cell. Changes in the membrane potential can lead to a variety of downstream effects, as they are tightly linked to such fundamental functions as cellular excitability, posttranslational modification of proteins by voltage-dependent phosphatases, hormone secretion, ion homeostasis, regulation of protein expression, and cell proliferation. A special situation exists in excitable cells (e.g., neurons or cardiomyocytes (CMs)), where relatively minor changes in the membrane potential can be amplified and result in chain-type activation of multiple ion channel types in a concerted manner, leading to generation of action potential. Even in non-excitable cells, changes in the membrane potential result in changes in electrochemical ion gradients and ion concentrations, which can trigger many seemingly voltage-independent processes.
Numerous in vitro applications that require advanced methods for manipulating the cell membrane potential include fundamental studies of molecular mechanisms in health and disease; functional probing of voltage-sensitive membrane proteins; studies of inter-cellular communications, including synaptic plasticity; cardiology-related projects; visual and olfactory transduction pathways; production and characterization of stem cell-derived cells for cell replacement therapy; activation-dependent maturation and differentiation of stem cell-derived cells; and high throughput screening of drug candidates to assess their potential cardiotoxicity and to support development of personalized medicine.
Novel methods for manipulating the cell membrane potential will also be extremely useful in in vivo applications under circumstances when there is a need to reinforce endogenous activation triggers that become impaired during the disease progression. For example, control over the membrane potential of CMs could help to overcome faulty electrical signaling, and thus, control abnormal heart rhythms. Degeneration of photoreceptors during retinitis pigmentosa or age-related macular degeneration can be compensated via light-driven electrical stimulation of next-in-line intact cells in the retina, potentially leading to restoration of visual perception. Stimulation of neurons can help to compensate for deficits in the neuronal activity in patients with cerebrovascular, neurodegenerative, and psychiatric diseases.
Electrophysiology is the most direct and precise method to control the cell membrane potential. Generally, electrophysiological stimulation by electrode-based techniques (e.g., patch pipettes, multielectrode arrays, or E-field stimulation) exhibits a high degree of control over the cell membrane potential. However, these techniques have significant drawbacks, such as limited, spatial selectivity, invasiveness, low throughput, poor amenability to automation, and general mechanical rigidity of current multi-electrode materials. Furthermore, electrode-based techniques are even less appropriate for studies of contracting CMs due to the damage that a stationary electrode causes in cells experiencing normal physiological movements.
Chemical methods to elicit changes in the membrane potential utilize such approaches as application of a “high K+” extracellular solution or pharmacological ion channel openers. Critical shortcomings of this method include the lack of control of the membrane potential, irreversibility of elicited changes, and low temporal and spatial resolution.
Optical stimulation methods utilize a light beam as an actuator, and thus, could allow long-term non-invasive control over the cell membrane potential. However, existing methods suffer from serious inherent shortcomings. One of the oldest methods in this category is light-triggered “uncaging” of chemically modified signaling molecules such as glutamate, ATP, dopamine, serotonin, cyclic nucleotides. Although the temporal resolution of this method is high, its spatial resolution is limited due to diffusion of “uncaged” molecules from the release site. Additionally, “caged” chemicals show precursor instability, phototoxicity, and require an intense UV illumination. Moreover, the “uncaging” process is irreversible and non-iterative.
The newest and the most promising optical stimulation method, optogenetics, is using genetically encoded transmembrane proteins with either inherent or engineered sensitivity to light. Optogenetics allows controlling the membrane potential in selected subpopulations of cells with unparalleled spatiotemporal precision. However, to provide the light-mediated control, optogenetics requires the expression of exogenous, functionally active proteins that become an integral part of the intracellular machinery. In other words, an investigator has to change physiology of a cell in order to be able to control its behavior, which could be detrimental in studies with stem-cell derived cells. Several technical challenges of optogenetics have already been addressed during a multi-iteration process of protein engineering and extensive tool development over the last several years. However, some problems are inherent to this approach: 1) Optogenetics does not provide physiological stimulation, as ion fluxes and initial membrane potential changes are determined by properties of exogenous proteins rather than endogenous ion channels. 2) High expression levels of light-sensitive proteins are required in order to achieve desired changes of the membrane potential. 3) In many case, addition of endogenous co-factor all-trans-retinal or photoisomerizable compounds is necessary for light-sensitive functioning of optogenetic proteins. 4) Optogenetics is not appropriate for pharmacological profiling of new drugs, because they can directly affect opsins instead or in addition to the target of interest.
Material Science: Photo-induced electrical excitation of neurons has been demonstrated using substrates made of inorganic bulk semiconductors, semiconductor nanoparticles, and organic semiconductor polymers. For example, neurons cultured on silicon wafers can be activated by light, because photo-induced changes in the conductivity of silicon in the presence of a specific voltage applied across the wafer can trigger a capacitive transient in neurons, resulting in their membrane depolarization. Unfortunately, 1) this platform is not transparent and, thus, has limited compatibility with optical detection methods; 2) very expensive and very rigid silicon wafers in a sophisticated controlling device drastically restrict its applicability; 3) the spatial resolution defined by the diameter of the photocurrent spreading is ≥50 μm. Some of these problems were addressed in a substrate where a photosensitive layer was made from a mixture of organic semiconducting polymers. Compared to a silicon-based substrate, this platform does not require any external devices, exhibits low heat dissipation, and has greater potential in terms of flexibility and fabrication simplicity. Its disadvantages include a) incompatibility with optical detection due to low transparency and the visible range excitation; and b) potential miniaturization problems due to mechanical brittleness of polymer layers. Another optical stimulation platform based on semiconductor nanoparticles provided the highest degree of engineering flexibility during its fabrication, but had the limited biocompatibility due to the presence of cadmium-containing components in nanoparticles.
In summary, novel technological tools are needed for remote stimulation of cells. Optimal physiological stimulation tools should be able to change the membrane potential quickly, reversibly, and repeatedly; to control the amplitude, duration, and direction of these changes; to be minimally intrusive and compatible with non-invasive detection methods.
To satisfy these requirements, novel materials with suitable properties can be considered. The family of graphene-based or graphene-related materials (here, the terms “grapliene-based” and “graphene-related” are used interchangeably) recently stepped into the spotlight after the 2010 Nobel Prize in Physics, and subsequent explosion in development of numerous applications for these materials in energy, electronics, sensors, light processing, medicine, and environmental fields. Graphene, the “founding” member of this family, is a two-dimensional material made of sp2-hybridized carbon atoms arranged in a hexagonal honeycomb lattice. The extended family of graphene-related materials includes graphene (single- and multi-layered), graphite, polycyclic aromatic hydrocarbons, carbon nanotubes, fullerenes, various graphene nanostructures of different dimensionalities (e.g., graphene nanoparticles, or graphene quantum dots: graphene nanoribbons: graphene nanomeshes; graphene nanodisks; graphene foams; graphene nanopillars), any combinations of other graphene-related materials, substituted graphene-related materials (e.g., the substitution of carbon atoms with N, B, P, S, Si, or others), and graphene-related materials functionalized with reactive functional groups (e.g., carboxyl groups, esters, amides, thiols, hydroxyl groups, diol groups, ketone groups, sulfonate groups, carbonyl groups, aryl groups, epoxy groups, phenol groups, phosphonic acids, amine groups, porphyrin, pyridine, polymers and combinations thereof). Specific examples of graphene-related materials include graphene oxide (GO), graphite oxide, and reduced graphene oxide (rGO).
Graphene and graphene-related materials exhibits extraordinary electronic, mechanical, and optical properties, which could make them invaluable in various biomedical applications, including construction of a biocompatible interface for remote stimulation of cells using electromagnetic radiation. Graphene is highly inert and chemically stable, which results in excellent biocompatibility. Its unique properties include high electrical conductivity, high mobilities of charge carriers, exceptional mechanical properties (e.g., high stiffness, strength, and elasticity), high thermal conductivity, broadband absorption and high transparency over the visible spectrum. Moreover, it is possible to tune electronic and optical properties of graphene for a specific application by pursuing one of many routes such as changing the stacking pattern of graphene sheets, changing the shape of graphene structures, substituting carbon atoms in a graphene lattice, and functionalizing graphene.
Graphene has a zero bandgap, leading to a broadband absorption of incident light with the energy below ˜3.5 eV. It means that graphene having the high optical absorption coefficient (7×105 cm−1) can efficiently detect wavelengths of light ranging from 300 to 2500 nm, which includes the entire visible spectrum, infrared and even terahertz regions.
The single layer of graphene is highly transparent with absorption of 2.3% across the visible spectrum and beyond with an absorption peak of ˜10% in the ultraviolet. When graphene absorbs photons, it transforms their energy into electrical current by creating photo-generated excitons via photoelectric and/or photo-thermoelectric mechanisms. The photoresponsivity of graphene is somewhat low (<10 mAW−1) due to the low optical absorption in monolayer graphene and the short recombination lifetime (on the scale of a picosecond) of the photo-generated carriers, leading to a low internal quantum efficiency of ˜6-16%.
GO is a highly oxidized graphene-related material with numerous oxygen-containing functional groups. In contrast to graphene with its zero band gap and high electronic conductivity, GO is a wide-bandgap semiconductor with a bandgap >3.5 eV, and very poor electronic conductivity. The removal of the oxygen-containing functional groups leads to the decrease in the optical bandgap from >3.5 eV to <1 eV, the increase in the optical absorption, and the restoration of the electrical conductivity. This reduction process results in another graphene-related material known as rGO or chemically converted graphene. While graphene and other non-functionalized graphene-related materials (e.g., graphite, carbon nanotubes and fullerenes), are hydrophobic, GO is hydrophilic due to the presence of oxygen-containing functional groups. rGO is intermediate in hydrophilicity because the number of remaining oxygen-containing functional groups in rGO is lower than in the highly oxidized GO.
Among current biomedical applications for graphene and its derivatives are their utilization for tissue engineering, antibacterial treatment, drug and gene delivery, and as contrast agents for bioimaging. For example, incorporation of graphene-related materials as structural elements both for planar or three-dimensional scaffolds for cell cultures greatly enhanced cell adhesion, improved the cell proliferation, accelerated the rate of cell maturation, enhanced the neurite sprouting and outgrowth, and supported the neuronal lineage during the stem cell differentiation. These applications utilized not only pure graphene and its derivatives, but also their hybrid nanocomposites with diverse nanostructures (e.g., semiconductor quantum dots, carbon nanotubes), proteins (e.g., chitosan), polymers (e.g., poly(propylene carbonate)), or other chemical entities (e.g., PEG). It was suggested that positive effects of graphene-related materials on creating favorable cell microenvironment are based on their mechanical properties, micro-scale topographic features, and surface chemistry characteristics.
All existing biomedical applications take advantage of passive steady-state properties of graphene and its derivatives. Currently, there are no applications that utilize external stimuli to actively change physicochemical properties of graphene-related biointerfaces, and subsequently to change the functional state of cells interacting with these interfaces.